Suppressing STAT3 activation impairs bone formation during maxillary expansion and relapse

Abstract Objectives The mid-palatal expansion technique is commonly used to correct maxillary constriction in dental clinics. However, there is a tendency for it to relapse, and the key molecules responsible for modulating bone formation remain elusive. Thus, this study aimed to investigate whether signal transducer and activator of transcription 3 (STAT3) activation contributes to osteoblast-mediated bone formation during palatal expansion and relapse. Methodology In total, 30 male Wistar rats were randomly allocated into Ctrl (control), E (expansion only), and E+Stattic (expansion plus STAT3-inhibitor, Stattic) groups. Micro-computed tomography, micromorphology staining, and immunohistochemistry of the mid-palatal suture were performed on days 7 and 14. In vitro cyclic tensile stress (10% magnitude, 0.5 Hz frequency, and 24 h duration) was applied to rat primary osteoblasts and Stattic was administered for STAT3 inhibition. The role of STAT3 in mechanical loading-induced osteoblasts was confirmed by alkaline phosphatase (ALP), alizarin red staining, and western blots. Results The E group showed greater arch width than the E+Stattic group after expansion. The differences between the two groups remained significant after relapse. We found active bone formation in the E group with increased expression of ALP, COL-I, and Runx2, although the expression of osteogenesis-related factors was downregulated in the E+stattic group. After STAT3 inhibition, expansive force-induced bone resorption was attenuated, as TRAP staining demonstrated. Furthermore, the administration of Stattic in vitro partially suppressed tensile stress-enhanced osteogenic markers in osteoblasts. Conclusions STAT3 inactivation reduced osteoblast-mediated bone formation during palatal expansion and post-expansion relapse, thus it may be a potential therapeutic target to treat force-induced bone formation.


Introduction
Insufficient maxilla width often leads to posterior crossbite and/or dental crowding. Treatment modalities, such as rapid maxillary expansion (RME), were implemented for correction. RME appliances subject the mid-palatal suture (MPS) to heavy forces, separating it and achieving new bone formation within this region. To ensure sufficient time for bone formation and maintain the stability of RME, a prolonged retention period, in which appliances are kept in situ for up to six to eight months is routinely conducted. However, poor patient cooperation, impaired oral hygiene, and appliance breakage due to prolonged retention are common in clinics, which could compromise RME treatment outcomes. Therefore, we first clarified tissue reconstruction in MPS during RME and post-expansion relapse by establishing a rat model. The effect of Stattic (a STAT3-specific inhibitor) on bone formation was further analyzed to elucidate the role of STAT3 in vivo. Since osteoblast-mediated bone formation is imperative in bone remodeling, we exposed rat primary osteoblasts to mechanical stretching and examined the involvement of STAT3.

Methodology
Animal specimens The study sample consisted of 30 male Wistar rats aged five weeks, with an average weight of 100±10 g.
The animals were randomly allocated to three groups (n=10): Ctrl (control; no expansion appliance), E (expansion only), and E+Stattic (expansion + STAT3 inhibition) groups. The experimental protocol was approved by the Animal Ethics Committee of West China Hospital of Stomatology of Sichuan University (WCHSIRB-D-2021-193).
A customized expansion device was fabricated as described previously 9,10 by bending a 0.014-inch Australian wire into a two-eye open spring. When the expander was inactivated, both arms were parallel ( Figure 1A). To activate the expander, the two arms were pulled aside symmetrically ( Figure 1B). Under anesthesia with a combination of ketamine (87 mg/kg) and xylazine (13 mg/kg), the expander was bonded onto the occlusal surfaces of rat molars with its two arms in parallel and exerting a force of 100±5 g ( Figure   1C). After 7-day expansion, the expanders were detached, and the rats experienced another 7-day relapse. Five rats from each group were euthanized on days seven (D7) 7 and 14 (D14).

Immunohistochemical staining
Immunohistochemical staining of COL-I, Runx2, and ALP was conducted as previously described.  According to the previously published study, 11 Image J was used to analyze ALP activity and alizarin red staining images quantitatively. Images were converted into "RGB stack" and the threshold was adjusted to identify mineralization areas. The ratio of mineralization areas was our readout.

Statistical analysis
Data were expressed as the mean ± standard deviation collected from three independent experiments. Inter-group comparisons were performed using Student's t-test (two groups) and one-way analysis of variance (three or more groups). Statistical significance was set at P<0.05 (version 7, GraphPad Software, Inc., La Jolla, CA, USA).

Results
STAT3 inactivation negatively affected the efficacy of palatal expansion and mid-palatal suture bone microstructure On D0, the baseline arch widths in all groups were consistent. After 1-week expansion, the E and E+Stattic groups showed increased arch width compared to that of the Ctrl group. The arch width of the E group was significantly greater than that of the E+Stattic group (p<0.05). For the E and E+Stattic groups, the arch width decreased after 1-week relapse.
However, the arch width of the E group at D14 was significantly greater than that of the other two groups

Enhanced bone remodeling induced by palatal expansion was attenuated by STAT3 inhibition
The intensity of osteogenic markers (ALP, Runx2, and COL-I) was examined using immunohistochemical staining. After 1-week expansion, the expression levels of ALP, Runx2, and COL-I were all upregulated. The inhibition of STAT3 partially reversed the increased expression of Runx2 and ALP, but their staining intensities were still higher than those of the Ctrl group. On D14, the expression of COL-I and Runx2 in the E group was greater than that in the Ctrl group.
No significant difference was found in the ALP staining intensity between each group on D14 ( Figure 6A-F).
TRAP staining was used in this study to demonstrate osteoclast activity. No obvious bone resorption was observed on D7 and D14 in the Ctrl group ( Figure   6G). In the E and E+Stattic groups, TRAP-positive Cyclic tensile stress-responsive calvarial osteoblasts showed higher expression of osteogenesis-related markers and mineralization capacity, and were attenuated by STAT3 inhibition First, the patterns of JAK2/STAT3 protein expression were assessed using western blotting. STAT3 protein expression was similar in all groups, whereas pSTAT3 expression increased after mechanical loading.
Moreover, JAK2, an upstream molecule of STAT3, was also phosphorylated by mechanical stimuli ( Figure   7A, B). Then, osteogenic molecule expression and mineralization capacity were examined. Tensile stress upregulated the expression of Runx2, COL-I, Osterix, and ALP, which was partially reversed by STAT3 inactivation (Figure 7C, D). The CTS group also exhibited increased ALP staining intensity and more mineralized nodules than the Ctrl group. However, Stattic administration decreased this promoting effect ( Figure 7E, F). These results confirmed that STAT3 modulated osteogenesis was enhanced by tensile stress. Discussion RME has been widely implemented by orthodontists for maxillary transverse deficiency in growing individuals. However, the high relapse rate due to insufficient bone formation/mineralization remains a prime concern. To minimize relapse, various interventions have been used in animal models to augment bone formation, such as treatment with parathyroid hormone, 14 Salvia officinalis L. extracts, 15 and salvianolic acid B. 16 These biological factors and drugs should stimulate bone formation in MPS subjected to RME. Thus, it is crucial to investigate the intrinsic mechanisms that drive bone formation. In the present study we identified that STAT3 participates in RME-induced bone formation by playing an anabolic  Finally, mechanical stimuli initiate bone formation and resorption. In this study, we primarily focused on the former. Future studies will need to delve deeper into the latter to provide a more complete picture.

Conclusions
Our results suggest that STAT3 inactivation impedes bone formation in the MPS during palatal expansion and post-expansion relapse. STAT3 is mechanosensitive in osteoblasts and can regulate its osteogenic response to mechanical stimuli. Therefore, STAT3 can be developed into a biotherapeutic drug target and requires further study.

Conflict of interest
The authors declare no conflict of interest.

Data availability statement
The datasets generated and analyzed during the current study are available in the SciELO